TSEs are a group of neurodegenerative diseases affecting a wide variety of mammals including sheep and goats (scrapie), cervid spp. (chronic wasting disease), and humans (Creutzfeldt-Jakob disease). Our studies are focused on the prion protein (PrP) due to the critical role of this protein in controlling many aspects of TSE pathogenesis such as susceptibility to disease and interspecies transmission. A central event in TSE disease involves the conversion of the normal host cellular prion protein (PrPC) to a partially protease-resistant, aggregated, disease-associated isoform (PrPSc). TSE-induced pathology is usually associated with PrP-res deposition, but the mechanism of neurodegeneration is not understood. The nature of the infectious agent, called a prion, remains uncertain but is thought to be composed primarily of misfolded PrP, perhaps in complex with another host accessory molecule(s). PrPC is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, and the majority of PrPSc produced in vivo contains this GPI anchor. Membrane association of both normal and disease-associated PrP isoforms may influence many features of prion disease and PrPC function. Our work is focused on elucidating mechanisms of uptake, replication, and spread of prions, in addition to determining the biochemical composition of mammalian prions and investigating factors that contribute to imparting the infectious phenotype to these prions, a unique feature among all protein misfolding diseases. [unreadable] [unreadable] Over the past year we have: 1) continued our characterization of how PrPSc is internalized and trafficked in neuronal cells by live cell imaging, expanding our studies to primary neuronal culture systems; 2) validated and optimized new methods to specifically tag PrPC molecules to visualize their trafficking in uninfected cells and during the course of scrapie infection; 3)applied these new methods to reveal new insights into the mechanism of action of an anti-TSE compound and trafficking pathways of PrPSc and PrPC; 4) created new compounds for protein labeling that allow analysis by a variety of detection methods; 5) developed new cell culture models expressing various PrPC mutant proteins; and 6) further characterized our novel cell culture models to visualize the trafficking of other modified prion and amyloid proteins by live cell imaging.